A program of study is proposed to determine the mechanism by which enzymes requiring 5'-deoxy-adenosylcobalamin (AdoCbl, coenzyme B12) as a cofactor accelerate the rate of homolysis of the carbon-cobalt bond of AdoCbl by 9.7 to 12 orders of magnitude. The enzyme to be studied is the ribonucleotide triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii which has recently been cloned and overexpressed in E. coli. This enzyme is unique among AdoCbl-dependent enzymes since it catalyzes the homolysis of AdoCbl without substrate, so that enzyme- induced homolysis can be studied in the absence of turnover. The kinetics of RTPR-induced AdoCbl homolysis will be studied as a function of temperature by stopped flow spectrophotometry in order to obtain values for the activation parameters for this process. Comparison to the activation parameters for the uncatalyzed homolysis of AdoCbl will permit a determination of the extent to which the enzyme catalyzes the reaction by lowering the enthalpy and/or by raising the entropy of activation. Molecular mechanics calculations and NMR studies of AdoCbl strongly suggest that at high temperatures, AdoCbl is substantially more conformationally flexible than it is at lower temperatures where the enzyme is active. As a result, it is believed that the activation parameters for AdoCbl measured at 85-110 degrees C are not appropriate for comparison to enzyme-induced homolysis at 37 degreesC Consequently, an initial rate method for studying the homolysis kinetics of AdoCbl at lower temperatures will be developed using very high specific activity [A2- 3H]AdoCbl tritiated at the adenosine C2 proton. Once the extent of enthalpic and entropic catalysis has been determined, a quantitative hypothesis for catalysis consisting of the following elements will be tested: (i) enzymatically induced upward flexing of the corrin ring to increase the steric restriction of acetamide side chain rotation by the Ado ligand (causing a decrease in ground state entropy and an increase in the entropy of activation) and possibly sterically stretching the Co-C bond by increased contact between the corrin ring nitrogens and the alpha methylene hydrogens of the Ado ligand, and (ii) stretching of the Co-C bond and bending of the Co-C-C bond angle by hydrogen bonding interactions between the active site and the Ado N7 and exocyclic amino groups, providing enthalpic catalysis. This hypothesis will be tested as follows: (l) Kinetic studies of the activation parameters for RTPR-induced homolysis of AdoCbl analogs with altered side chain and Ado ligand structure. (2) 15N and 15N-edited 1H NMR studies of [U-15N]AdoCbl (from fermentation) complexed to RTPR to probe hydrogen bonding interactions of the active site with the Ado ligand and with the side chain amides. (3) 13C NMR studies of [A15-13C]AdoCbl (labeled at the cobalt-bound carbon) complexed to RTPR to look for evidence of ground state Co-C bond strain. (4) Development of NMR probes of corrin conformation, including the use of 13C chemical shifts as an indicator of corrin ring fold and NOE constrained molecular mechanics calculations, to permit determination of corrin ring conformation in complexes of [U- 13C]AdoCbl (from fermentation) with RTPR.